Compositions, their manufacture and use

ABSTRACT

Composition containing (A) at least one salt of an organic ester of choline or of a derivative of choline, and (B) at least one surfactant selected from amphoteric and anionic and non-ionic surfactants.

The present invention is directed towards a composition containing

-   -   (A) at least one salt according to general formula (1)

(R²)₃N⁺—(CH₂)_(n)C(R³)(R⁴)—(O—X)_(m)O—C(O)—R¹ A⁻  (1)

-   -   -   wherein         -   n is selected from 1 to 12,         -   m is selected from zero to 50,         -   R¹ is selected from C₁-C₁₀-alkyl, linear or branched, and             C₆-C₁₀-aryl, wherein R¹ may bear one or more hydroxyl or C═O             or COOH groups, partially or fully neutralized, if             applicable,         -   R² are same or different and selected from C₁-C₁₀-alkyl,             phenyl,         -   R³ and R⁴ are same or different and selected from hydrogen             and C₁-C₄-alkyl,         -   X is C₂-C₄-alkylen, and         -   A⁻ is a counteranion, inorganic or organic, and

    -   (B) at least one surfactant selected from amphoteric and anionic         and non-ionic surfactants.

Compositions and especially detergent compositions are made for various cleaning applications, for example laundry or automatic dishwashing. All of them have to fulfil many different requirements. While solid compositions play a traditional role, in the meantime liquid compositions also have their merits. They can be completely transferred into a laundromat without leaving residues of unused detergent in the dosage unit. Especially for so-called color laundry detergents, liquid laundry detergents are of great importance.

Detergent manufacturer demand a great flexibility from the ingredients they use, including the builders. With the growing popularity of liquid heavy duty detergents, a well-known builder is sodium citrate, the trisodium salt of citric acid. Sodium citrate is suitable for use as a builder in heavy duty laundry detergents because of its ability to sequester calcium and magnesium ions found in tap water and, unlike phosphate builders, it is environmentally safe. It is especially suitable for use in liquid detergent formulations because, unlike other environmentally safe detergent builders, trisodium citrate is well soluble. A minimum water content of 30% by weight is usually required, though. This requirement limits the flexibility of the laundry detergent manufacturers. This requirement turns out to be even more detrimental in view of the modern trend within this industry, namely downsizing of dosage units (pouches) and therefore reducing the water content.

One additional problem that laundry detergents face is the shelf life. Especially liquid compositions usually lose their activity after some time. Various reasons have been identified. One component that may be deactivated after some time are enzymes. While enzymes are useful components for, e.g., removal of foods stains like egg, fruit and the like, their activity deteriorated after some time on shelf or at the end-user. Boron compounds that are known as enzyme stabilizers, see, e.g., U.S. Pat. No. 4,465,619, are now under scrutiny.

In WO 97/03156, certain choline derivatives and their use as surfactants are disclosed.

It was therefore an objective of the present invention to provide a composition that allows a flexible formulation, even with high or low water content. It was further the objective to provide a composition that has excellent shelf life, especially with respect to the enzyme(s) contained therein. It was further an objective to provide a method for making such compositions, and it was further an objective to provide uses of such compositions.

Accordingly, the compositions as defined at the outset have been found, hereinafter also defined as inventive compositions or as compositions according to the present invention. Inventive compositions contain

-   -   (A) at least one salt according to general formula (I),         hereinafter altogether also referred to as ester (A) or salt (A)         or component (A), and     -   (B) at least one surfactant selected from amphoteric and anionic         and non-ionic surfactants, hereinafter also referred to as         surfactant (B) or component (B).

Salt (A) is a salt of an organic ester of choline or of a derivative of choline. The anion of salt (A)—the counterion—may be inorganic or organic, organic being preferred. Examples of inorganic counterions of salt (A) are nitrate, hydroxide, sulphate, phosphate, hydrogenphosphate, dihydrogenphosphate, carbonate, bicarbonate, and halide, for example bromide or chloride. Preferred are halide, especially chloride, and sulphate, carbonate, and bicarbonate. Examples of organic counterions are lactate, acetate, tartrate, citrate, and CH₃SO₃ ⁻ (methanesulfonate). In embodiments with divalent or trivalent counterions, the respective molar amounts cation is present.

The nitrogen atom in salt (A) bears three methyl groups and a hydroxyethyl group. The term derivatives of choline as used in the context of the present invention refers to compounds that bear at least one alkyl group other than a methyl group, or a hydroxyalkyl group other than a 2-hydroxyethyl group, or further alkoxy groups.

More specifically, salt (A) is a compound of general formula (1)

(R²)₃N⁺—(CH₂)_(n)C(R³)(R⁴)—(O—X)_(m)O—C(O)—R¹ A⁻  (1)

wherein

n is selected from 1 to 12, for example 1 to 9, preferably 1, 2, 3, or 4, and even more preferably n is 1,

m is selected from zero to 50, for example 2 to 50, preferred is 10 to 25. Most preferably, however, m is zero.

R¹ is selected from C₁-C₁₀-alkyl, linear or branched, and C₆-C₁₀-aryl, wherein R¹ may bear one or more hydroxyl or C═O or COOH groups, partially or fully neutralized, if applicable. Preferred examples of R¹ are non-substituted C₁-C₆-alkyl such as methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, n-hexyl, preferred non-substituted C₁-C₁₀-alkyl are methyl and ethyl, furthermore substituted such C₁-C₁₀-alkyl as —CH(OH)—CH(OH)—COOH, CH(OH)—CH₃, (E)-CH═CHCOOH, (Z)—CH═CHCOOH, —C₆H₅, para-HO—C₆H₄—, o,p-dihydroxyphenyl, and 3,4,5-triydroxyphenyl. In a preferred embodiment, O—C(O)—R¹ together constitute a citrate. Even more preferred, R¹ is methyl.

R² are same or different and selected from phenyl and C₁-C₁₀-alkyl, for example methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec.-butyl, tert.-butyl, n-pentyl, iso-pentyl, sec.-pentyl, neo-pentyl, 1,2-dimethylpropyl, iso-amyl, n-hexyl, iso-hexyl, sec.-hexyl, n-heptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, 2-n-propyl-heptyl, or iso-decyl, preferred are linear C₁-C₁₀-alkyl and more preferred are linear C₁-C₄-alkyl, Even more preferred at least two R² groups are CH₃ and the third R² is selected from linear C₁-C₁₀-alkyl, and most preferred, all R² are the same and methyl.

R³ and R⁴ are same or different and selected from hydrogen and C₁-C₄-alkyl, preferred are n- for example methyl, ethyl, n-propyl, and n-butyl, and even more preferred both R³ and R⁴ are hydrogen. In another embodiment, R³ is C₁-C₄-alkyl and R⁴ is hydrogen, preferably R³ is methyl and R⁴ is hydrogen.

X is C₂-C₄-alkylen, for example —CH₂—CH₂—, —CH(CH₃)—CH₂—, —(CH₂)₃—, CH₂—CH(CH₃)—, or —(CH₂)₄—, and

A⁻ is a counteranion, inorganic or organic. Examples of inorganic counterions of salt (A) are sulphate, phosphate, hydrogenphosphate, dihydrogenphosphate, carbonate, bicarbonate, and halide, for example bromide or chloride. Preferred are halide, especially chloride, and sulphate, carbonate, and bicarbonate. Examples of organic counterions are lactate, acetate, tartrate, citrate, and CH₃SO₃ ⁻ (methanesulfonate).

Most preferred example of salts (A) are salts of choline esters with tartrate or citrate as counterion, and salts of acetyl choline, choline citrate, and of choline tartrate, for example lactates, acetates, tartrates, citrates.

Component (a) is not a surfactant. In one embodiment, a solution of 5 g component (a) in 1000 g water has a dynamic surface tension of >45 mN/m at 20° C. and 101.3 kPa. A solution of 5 g component (a) in 1000 g water may have a dynamic surface tension of >50 mN/m at 20° C. and 101.3 kPa. The dynamic surface tension may be measured with a bubble pressure tensiometer, wherein the maximum internal pressure of a gas bubble which is formed in a liquid by means of a capillary is measured; the measured value usually corresponds to the surface tension at a certain surface age, the time from the start of the bubble formation to the occurrence of the pressure maximum. In one embodiment, the surface age during measuring the dynamic surface tension at 20° C. and 101.3 kPa with a bubble pressure tensiometer is 50 ms.

In embodiments wherein counteranion A⁻ is—or may be—divalent such as sulphate, tartrate, carbonate, or polyvalent such as phosphate or citrate, the necessary positive charge may be furnished by another salt (A) derived cation, or by alkali metal cations such as potassium or preferably sodium, or by ammonium, non-substituted or substituted with C₁-C₄-alkyl and/or with 2-hydroxyethyl.

In embodiments wherein R¹ bears one or more carboxyl groups they may be free COOH groups or partially or fully neutralized with alkali, for example potassium or especially sodium, or they may be esterified, for example with (R²)₃N⁺—(CH₂)_(n)—(O—X)_(m)OH. Such embodiments result in the di- or triester, if applicable, of the respective di- or tricarboxylic acid. Mixtures of mono- and diesters of, e.g., tartaric acid or citric acid, and mixtures of di- and triesters of citric acid are feasible as well.

In a preferred embodiment of the present invention, salt (A) is selected from

(CH₃)₃N⁺—(CH₂)₂—O—C(O)—R⁵ (A¹)⁻  (II)

wherein (A¹)⁻ is selected from methanesulfonate, tartrate and citrate and wherein R⁵ is selected from —CH₂—C(OH)(COOX²)—CH₂—COOX² and —CH(OH)—CH(OH)—COOX¹

wherein X¹ is selected from hydrogen, alkali metal—especially sodium—and (CH₃)₃N⁺—(CH₂)₂— and wherein X² are same or different and selected from hydrogen, alkali metal—especially sodium—and (CH₃)₃N⁺—(CH₂)₂—. In a preferred embodiment, the ester group of O—C(O)—R⁵ and (A¹)⁻ correspond to each other.

In one embodiment of the present invention, inventive compositions contain in the range of from 0.5 to 30% by weight of salt (A), referring to the entire composition, preferably 0.5 to 6% by weight and more preferably 1 to 3% by weight.

In one embodiment of the present invention, salt (A) contains as an impurity a compound (A′)

(R²)₃N⁺—(CH₂)_(n)C(R³)(R⁴)—(O—X)_(m)OH R¹—COO—

wherein the variables R¹, R², X, n and m are the same as in the corresponding salt (A). Said impurity may amount to up to 50 mole-%, preferably 0.1 to 20 mole-%, even more preferably 1 to 10 mole-% of salt (A). Although impurity (A′) may stem from the synthesis of salt (A) and may be removed by purification methods it is not preferred to remove it.

Inventive compositions further contain at least one surfactant (B) selected from amphoteric surfactants and especially from anionic surfactants and non-ionic surfactants, hereinafter also referred to as amphoteric surfactants (B), anionic surfactants (B) and non-ionic surfactants (B), respectively.

Examples of suitable anionic surfactants (B) are alkali metal and ammonium salts of C₈-C₁₈-alkyl sulfates, of C₈-C₁₈-fatty alcohol polyether sulfates, of sulfuric acid half-esters of ethoxylated C₄-C₁₂-alkylphenols (ethoxylation: 1 to 50 mol of ethylene oxide/mol), C₁₂-C₁₈ sulfo fatty acid alkyl esters, for example of C₁₂-C₁₈ sulfo fatty acid methyl esters, furthermore of C₁₂-C₁₈-alkylsulfonic acids and of C₁₀-C₁₈-alkylarylsulfonic acids. Preference is given to the alkali metal salts of the aforementioned compounds, particularly preferably the sodium salts.

Specific examples of anionic surfactants (B) are compounds according to general formula (III)

C_(s)H_(2s+1)—O(CH₂CH₂O)_(t)—SO₃M  (III)

wherein

-   s being a number in the range of from 10 to 18, preferably 12 to 14,     and even more preferably s=12, -   t being a number in the range of from 1 to 5, preferably 2 to 4 and     even more preferably 3. -   M being selected from alkali metals, preferably potassium and even     more preferably sodium.

In surfactant (B), the variables s and t may be average numbers and therefore they are not necessarily whole numbers, while in individual molecules according to formula (III), both s and t denote whole numbers.

Further examples for suitable anionic surfactants (B) are soaps, for example the sodium or potassium salts of stearic acid, oleic acid, palmitic acid, ether carboxylates, and alkylether phosphates.

Preferred non-ionic surfactants (B) are alkoxylated alcohols, di- and multiblock copolymers of ethylene oxide and propylene oxide and reaction products of sorbitan with ethylene oxide or propylene oxide, alkyl polyglycosides (APG), hydroxyalkyl mixed ethers and amine oxides.

Preferred examples of alkoxylated alcohols and alkoxylated fatty alcohols are, for example, compounds of the general formula (IV)

wherein

-   R⁶ is identical or different and selected from hydrogen and linear     C₁-C₁₀-alkyl, preferably in each case identical and ethyl and     particularly preferably hydrogen or methyl, -   R⁷ is selected from C₈-C₂₂-alkyl, branched or linear, for example     n-C₈H₁₇, n-C₁H₂₁, n-C₁₂H₂₅, n-C₁₄H₂₉, n-C₁₆H₃₃ or n-C₁₈H₃₇, -   R⁸ is selected from C₁-C₁₀-alkyl, methyl, ethyl, n-propyl,     isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,     isopentyl, sec-pentyl, neopentyl, 1,2-dimethylpropyl, isoamyl,     n-hexyl, isohexyl, sec-hexyl, n-heptyl, n-octyl, 2-ethylhexyl,     n-nonyl, n-decyl or isodecyl.

The variables m and x are in the range from zero to 300, where the sum of n and m is at least one, preferably in the range of from 3 to 50. Preferably, m is in the range from 1 to 100 and x is in the range from 0 to 30.

In one embodiment, compounds of the general formula (III) may be block copolymers or random copolymers, preference being given to block copolymers.

Other preferred examples of alkoxylated alcohols are, for example, compounds of the general formula (V)

wherein

-   R⁶ is identical or different and selected from hydrogen and linear     C₁-C₁₀-alkyl, preferably identical in each case and ethyl and     particularly preferably hydrogen or methyl, R⁹ is selected from     C₆-C₂₀-alkyl, branched or linear, in particular n-C₈H₁₇, n-C₁H₂₁,     n-C₁₂H₂₅, n-C₁₃H₂₇, n-C₁₅H₃₁, n-C₁₄H₂₉, n-C₁₆H₃₃, n-C₁₈H₃₇, -   a is a number in the range from zero to 10, preferably from 1 to 6, -   b is a number in the range from 1 to 80, preferably from 4 to 20, -   d is a number in the range from zero to 50, preferably 4 to 25.

The sum a+b+d is preferably in the range of from 5 to 100, even more preferably in the range of from 9 to 50.

Preferred examples for hydroxyalkyl mixed ethers are compounds of the general formula (VI)

in which the variables are defined as follows:

-   R⁶ is identical or different and selected from hydrogen and linear     C₁-C₁₀-alkyl, preferably in each case identical and ethyl and     particularly preferably hydrogen or methyl, -   R⁷ is selected from C₈-C₂₂-alkyl, branched or linear, for example     iso-C₁₁H₂₃, iso-C₁₃H₂₇, n-C₈H₁₇, n-C₁₀H₂₁, n-C₁₂H₂₅, n-C₁₄H₂₉,     n-C₁₆H₃₃ or n-C₁₈H₃₇, -   R⁸ is selected from C₁-C₁₈-alkyl, methyl, ethyl, n-propyl,     isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,     isopentyl, sec-pentyl, neopentyl, 1,2-dimethylpropyl, isoamyl,     n-hexyl, isohexyl, sec-hexyl, n-heptyl, n-octyl, 2-ethylhexyl,     n-nonyl, n-decyl, isodecyl, n-dodecyl, n-tetradecyl, n-hexadecyl,     and n-octadecyl.

The variables m and x are in the range from zero to 300, where the sum of n and m is at least one, preferably in the range of from 5 to 50. Preferably, m is in the range from 1 to 100 and n is in the range from 0 to 30.

Compounds of the general formulae (V) and (VI) may be block copolymers or random copolymers, preference being given to block copolymers.

Further suitable non-ionic surfactants are selected from di- and multiblock copolymers, composed of ethylene oxide and propylene oxide. Further suitable non-ionic surfactants are selected from ethoxylated or propoxylated sorbitan esters. Amine oxides or alkyl polyglycosides, especially linear C₄-C₁₈-alkyl polyglucosides and branched C₈-C₁₈-alkyl polyglycosides such as compounds of general average formula (VII) are likewise suitable.

wherein:

-   R¹⁰ is C₁-C₄-alkyl, in particular ethyl, n-propyl or isopropyl, -   R¹¹ is —(CH₂)₂—R¹⁰, -   G¹ is selected from monosaccharides with 4 to 6 carbon atoms,     especially from glucose and xylose, -   y in the range of from 1.1 to 4, y being an average number.

Further examples of non-ionic surfactants are compounds of general formula (VIII) and (IX)

-   AO is selected from ethylene oxide, propylene oxide and butylene     oxide, -   EO is ethylene oxide, CH₂CH₂—O, -   R¹⁰ is C₁-C₄-alkyl, in particular ethyl, n-propyl or isopropyl, -   R¹² selected from C₈-C₁₈-alkyl, branched or linear -   A³O is selected from propylene oxide and butylene oxide, -   w is a number in the range of from 15 to 70, preferably 30 to 50, -   w1 and w3 are numbers in the range of from 1 to 5, and -   w2 is a number in the range of from 13 to 35.

An overview of suitable further non-ionic surfactants can be found in EP-A 0 851 023 and in DE-A19819187.

Mixtures of two or more different non-ionic surfactants (B) may also be present.

Other surfactants that may be present are selected from amphoteric (zwitterionic) surfactants and anionic surfactants and mixtures thereof.

Examples of amphoteric surfactants (B) are those that bear a positive and a negative charge in the same molecule under use conditions. Preferred examples of amphoteric surfactants are so-called betaine-surfactants. Many examples of betaine-surfactants bear one quaternized nitrogen atom and one carboxylic acid group per molecule. A particularly preferred example of amphoteric surfactants is cocamidopropyl betaine (lauramidopropyl betaine).

Examples of amine oxide surfactants are compounds of the general formula (X)

R¹³R¹⁴R¹⁵N→O  (X)

wherein R¹³, R¹⁴ and R¹⁵ are selected independently from each other from aliphatic, cycloaliphatic or C₂-C₄-alkylene C₁₀-C₂₀-alkylamido moieties. Preferably, R¹² is selected from C₈-C₂₀-alkyl or C₂-C₄-alkylene C₁₀-C₂₀-alkylamido and R¹³ and R¹⁴ are both methyl.

A particularly preferred example is lauryl dimethyl aminoxide, sometimes also called lauramine oxide. A further particularly preferred example is cocamidylpropyl dimethylaminoxide, sometimes also called cocamidopropylamine oxide.

In one embodiment of the present invention, inventive compositions contain 0.1 to 60% by weight of surfactant (B), preferably selected from anionic surfactants (B), non-ionic surfactants (B), amphoteric surfactants (B) and amine oxide surfactants as well as combinations of at least two of the foregoing. In a preferred embodiment, inventive compositions contain 5 to 30% by weight of anionic surfactant and at least one non-ionic surfactant, for example in the range of from 3 to 20% by weight.

Inventive compositions may be liquid at ambient temperature (20° C.). Preferably, they are stable liquids at ambient temperature. In this context, the term “stable” means that such inventive compositions are liquid and do not show traces of precipitate formation or turbidity even after 20 days of storage.

In one embodiment of the present invention, inventive compositions are aqueous, typically containing in the range of from 5 to 95% by weight water, for example 5 to 30% by weight and more preferred 5 to 25% by weight, or 20 to 70% by weight water and zero to 20% by weight organic solvent. Preferred is 5 to 15% by weight of water and no significant amounts of organic solvent, for example 1% by weight or less.

Inventive compositions may be alkaline or exhibit a neutral or slightly acidic pH value, for example 6 to 14, preferably 6.5 to 13, more preferably 8 to 10.5 and most preferably 8.5 to 9.0.

In one embodiment of the present invention, inventive compositions additionally comprise

-   -   (C) at least one enzyme, hereinafter also referred to as enzyme         (C), for example hydrolase (C). Preferred enzymes (C) are         selected from proteases, amylases, and lipases, hereinafter also         referred to as proteases (C), amylases (C), or lipases (C),         respectively.

It has been found that enzymes (C) in inventive compositions exhibit a particularly high life-time. Enzymes (C) that are particularly beneficial in inventive compositions are selected from hydrolases (EC 3). Preferred enzymes (C) are selected from the group of enzymes acting on ester bond (E.C. 3.1), glycosylases (E.C. 3.2), and peptidases (E.C. 3.4). Enzymes acting on ester bond (E.C. 3.1), hereinafter also refer to lipases (component (b)), respectively. Glycosylases (E.C. 3.2) hereinafter also refer to either amylases (C) and cellulases (C). Peptidases hereinafter also refer to proteases (C).

Hydrolases (C) in the context of the present invention are identified by polypeptide sequences, also called amino acid sequences herein. The polypeptide sequence specifies the three-dimensional structure including the “active site” of an enzyme which in turn determines the catalytic activity of the same. Polypeptide sequences may be identified by a SEQ ID NO. According to the World Intellectual Property Office (WIPO) Standard ST.25 (1998) the amino acids herein are represented using three-letter code with the first letter as a capital or the corresponding one letter.

Enzymes (C) in the context of the present invention may relate to parent enzymes and/or variant enzymes, both having enzymatic activity. Enzymes having enzymatic activity are enzymatically active or exert enzymatic conversion, meaning that enzymes act on substrates and convert these into products. The term “enzyme” herein excludes inactive variants of an enzyme.

A “parent” sequence of a parent protein or enzyme, also called “parent enzyme” is the starting sequence for introduction of changes, e.g., by introducing one or more amino acid substitutions, insertions, deletions, or a combination thereof to the sequence, resulting in “variants” of the parent sequences. The term parent enzyme (or parent sequence) includes wild-type enzymes (sequences) and synthetically generated sequences (enzymes) which are used as starting sequences for introduction of (further) changes.

The term “enzyme variant” or “sequence variant” or “variant enzyme” refers to an enzyme that differs from its parent enzyme in its amino acid sequence to a certain extent. If not indicated otherwise, variant enzyme “having enzymatic activity” means that this variant enzyme has the same type of enzymatic activity as the respective parent enzyme.

In describing the variants of the present invention, the nomenclature described as follows is used:

Amino acid substitutions are described by providing the original amino acid of the parent enzyme followed by the number of the position within the amino acid sequence, followed by the substituted amino acid.

Amino acid deletions are described by providing the original amino acid of the parent enzyme followed by the number of the position within the amino acid sequence, followed by *.

Amino acid insertions are described by providing the original amino acid of the parent enzyme followed by the number of the position within the amino acid sequence, followed by the original amino acid and the additional amino acid. For example, an insertion at position 180 of lysine next to glycine is designated as “Gly180GlyLys” or “G180GK”.

In cases where a substitution and an insertion occur at the same position, this may be indicated as S99SD+S99A or in short S99AD. In cases where an amino acid residue identical to the existing amino acid residue is inserted, it is clear that degeneracy in the nomenclature arises. If for example a glycine is inserted after the glycine in the above example this would be indicated by G180GG.

Where different alterations can be introduced at a position, the different alterations are separated by a comma, e.g. “Arg170Tyr, Glu” represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Alternatively different alterations or optional substitutions may be indicated in brackets e.g. Arg170[Tyr, Gly] or Arg170{Tyr, Gly}; or in short R170 [Y,G] or R170 {Y, G}; or in long R170Y, R170G.

Enzyme variants may be defined by their sequence identity when compared to a parent enzyme. Sequence identity usually is provided as “% sequence identity” or “% identity”. For calculation of sequence identities, in a first step a sequence alignment has to be produced. According to this invention, a pairwise global alignment has to be produced, meaning that two sequences have to be aligned over their complete length, which is usually produced by using a mathematical approach, called alignment algorithm.

In the context of the present invention, the alignment is generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. 1979 48, p. 443-453). Preferably, the program “NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) is used for the purposes of the current invention, with using the programs default parameter (gap open=10.0, gap extend=0.5 and matrix=EBLOSUM62). According to this invention, the following calculation of %-identity applies: %-identity=(identical residues/length of the alignment region which is showing the respective sequence of this invention over its complete length)·100.

In the context of the present invention, enzyme variants may be described as an amino acid sequence which is at least n % identical to the amino acid sequence of the respective parent enzyme with “n” being an integer between 10 and 100. In one embodiment, variant enzymes are at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical when compared to the full length amino acid sequence of the parent enzyme, wherein the enzyme variant has enzymatic activity.

Enzyme variants may be defined by their sequence similarity when compared to a parent enzyme. Sequence similarity usually is provided as “% sequence similarity” or “%-similarity”. % sequence similarity takes into account that defined sets of amino acids share similar properties, e.g by their size, by their hydrophobicity, by their charge, or by other characteristics. Herein, the exchange of one amino acid with a similar amino acid may be called “conservative mutation”.

For determination of %-similarity according to this invention the following applies: amino acid A is similar to amino acids S; amino acid D is similar to amino acids E and N; amino acid E is similar to amino acids D and K and Q; amino acid F is similar to amino acids W and Y; amino acid H is similar to amino acids N and Y; amino acid I is similar to amino acids L and M and V; amino acid K is similar to amino acids E and Q and R; amino acid L is similar to amino acids I and M and V; amino acid M is similar to amino acids I and L and V; amino acid N is similar to amino acids D and H and S; amino acid Q is similar to amino acids E and K and R; amino acid R is similar to amino acids K and Q; amino acid S is similar to amino acids A and N and T; amino acid T is similar to amino acids S; amino acid V is similar to amino acids I and L and M; amino acid W is similar to amino acids F and Y; amino acid Y is similar to amino acids F and H and W.

Conservative amino acid substitutions may occur over the full length of the sequence of a polypeptide sequence of a functional protein such as an enzyme. In one embodiment, such mutations are not pertaining the functional domains of an enzyme. In one embodiment, conservative mutations are not pertaining the catalytic centers of an enzyme.

To take conservative mutations into account, a value for sequence similarity of two amino acid sequences may be calculated from the same alignment, which is used to calculate %-identity. According to this invention, the following calculation of %-similarity applies: %-similarity=[(identical residues+similar residues)/length of the alignment region which is showing the respective sequence(s) of this invention over its complete length]-100.

In the context of the present invention, enzyme variants may be described as an amino acid sequence which is at least m % similar to the respective parent sequences with “m” being an integer between 10 and 100. In one embodiment, variant enzymes are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar when compared to the full length polypeptide sequence of the parent enzyme, wherein the variant enzyme has enzymatic activity.

“Enzymatic activity” means the catalytic effect exerted by an enzyme, which usually is expressed as units per milligram of enzyme (specific activity) which relates to molecules of substrate transformed per minute per molecule of enzyme (molecular activity).

Variant enzymes may have enzymatic activity according to the present invention when said enzyme variants exhibit at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at 10 least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the enzymatic activity of the respective parent enzyme.

In one embodiment, inventive compositions comprise at least one protease (C). Enzymes (C) having proteolytic activity are called “proteases” or peptidases in the context of the present invention. Such enzymes are members of class EC 3.4.

Proteases (C) are further classified as aminopeptidases (EC 3.4.11), dipeptidases (EC 3.4.13), dipeptidyl-peptidases and tripeptidyl-peptidases (EC 3.4.14), peptidyl-dipeptidases (EC 3.4.15), serine-type carboxypeptidases (EC 3.4.16), metallocarboxypeptidases (EC 3.4.17), cysteinetype carboxypeptidases (EC 3.4.18), omega peptidases (EC 3.4.19), serine endopeptidases (EC 3.4.21), cysteine endopeptidases (EC 3.4.22), aspartic endopeptidases (EC 3.4.23), metallo-endopeptidases (EC 3.4.24), threonine endopeptidases (EC 3.4.25), or endopeptidases of unknown catalytic mechanism (EC 3.4.99).

Protease (C) may be an endopeptidase of any kind or a mixture of endopeptidases of any kind. In one embodiment, protease according to the invention is selected from serine protease (EC 3.4.21).

Serine proteases or serine peptidases are characterized by having a serine in the catalytically active site, which forms a covalent adduct with the substrate during the catalytic reaction. A serine protease in the context of the present invention is selected from the group consisting of chymotrypsin (e.g., EC 3.4.21.1), elastase (e.g., EC 3.4.21.36), elastase (e.g., EC 3.4.21.37 or EC 3.4.21.71), granzyme (e.g., EC 3.4.21.78 or EC 3.4.21.79), kallikrein (e.g., EC 3.4.21.34, EC 3.4.21.35, EC 3.4.21.118, or EC 3.4.21.119,) plasmin (e.g., EC 3.4.21.7), trypsin (e.g., EC 3.4.21.4), thrombin (e.g., EC 3.4.21.5), and subtilisin. Subtilisin is also known as subtilopeptidase, e.g., EC 3.4.21.62, the latter hereinafter also being referred to as “subtilisin”.

Crystallographic structures of proteases (C) show that the active site is commonly located in a groove on the surface of the molecule between adjacent structural domains, and the substrate specificity is governed by the properties of binding sites arranged along the groove on one or both sides of the catalytic site that is responsible for hydrolysis of the scissile bond. Accordingly, the specificity of protease (C) can be described by use of a conceptual model in which each specificity subsite is able to accommodate the sidechain of a single amino acid residue. The sites are numbered from the catalytic site, S1, S2 . . . Sn towards the N-terminus of the substrate, and S1′, S2′ . . . Sn′ towards the C-terminus. The residues they accommodate are numbered P1, P2 . . . Pn, and P1′, P2′ . . . Pn′, respectively:

Substrate P3 P2 P1 + P1′ P2′ P3′ Enzyme S3 S2 S1 * S1′ S2′ S3′

In this representation the catalytic site of the enzyme is marked “*” and the peptide bond cleaved (the scissile bond) is indicated by the symbol “+”.

In general, the three main types of protease activity (proteolytic activity) are: trypsin-like, where there is cleavage of amide substrates following Arg (N) or Lys (K) at P1, chymotrypsin-like where cleavage occurs following one of the hydrophobic amino acids at P1, and elastase-like with cleavage following an Ala (A) at P1.

A sub-group of the serine proteases tentatively designated as subtilases has been proposed by Siezen et al. (1991), Protein Eng. 4:719-737 and Siezen et al. (1997), Protein Science 6:501-523. They are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously often defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases. A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases has been determined. For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al. (1997), Protein Science 6:501-523. Subtilases may be divided into 6 sub-divisions, i.e. the subtilisin family, the thermitase family, the proteinase K family, the lantibiotic peptidase family, the kexin family and the pyrolysin family.

A subgroup of the subtilases are the subtilisins which are serine proteases from the family S8 as defined by the MEROPS database (http://merops.sanger.ac.uk). Peptidase family S8 contains the serine endopeptidase subtilisin and its homologues. In subfamily S8A, the active site residues frequently occur in the motifs Asp-Thr/Ser-Gly similarly to the sequence motif in families of aspartic endopeptidases in clan AA, His-Gly-Thr-His and Gly-Thr-Ser-Met-Ala-Xaa-Pro.

Prominent members of family S8, subfamily A are:

MEROPS Family name S8, Subfamily A Subtilisin Carlsberg S08.001 Subtilisin lentus S08.003 Thermitase S08.007 Subtilisin BPN' S08.034 Subtilisin DY S08.037 Alkaline peptidase S08.038 Subtilisin ALP 1 S08.045 Subtilisin sendai S08.098 Alkaline elastase YaB S08.157

The subtilisin related class of serine proteases shares a common amino acid sequence defining a catalytic triad which distinguishes them from the chymotrypsin related class of serine proteases. Subtilisins and chymotrypsin related serine proteases both have a catalytic triad comprising aspartate, histidine and serine.

In subtilisin related proteases (C) the relative order of these amino acids, reading from the amino to carboxy-terminus is aspartate-histidine-serine. In the chymotrypsin related proteases the relative order, however is histidine-aspartate-serine. Thus, subtilisin herein refers to a serine protease having the catalytic triad of subtilisin related proteases. Examples include the subtilisins as described in WO 89/06276 and EP 0283075, WO 89/06279, WO 89/09830, WO 89/09819, WO 91/06637 and WO 91/02792.

Parent proteases of the subtilisin type (EC 3.4.21.62) and variants may be bacterial proteases. Said bacterial protease may be a Gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces protease, or a Gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma protease. They act as unspecific endopeptidases, i.e. they hydrolyze any peptide bonds.

Commercially available protease enzymes include those sold under the trade names Alcalase®, Blaze®, Duralase™, Duraym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase® and Esperase® (Novozymes A/S), those sold under the tradename Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect® Prime, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax, Properase®, FN2®, FN3®, FN4®, Excellase®, Eraser®, Ultimase®, Opticlean®, Effectenz®, Preferenz® and Optimase (Danisco/DuPont), Axapem™ (Gist-Brocases N.V.), Bacillus lentus Alkaline Protease (BLAP; sequence shown in FIG. 29 of U.S. Pat. No. 5,352,604) and variants thereof and KAP (Bacillus alkalophilus subtilisin) from Kao.

In one aspect of the invention, the parent enzymes and variants may be a Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus gibsonii, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis protease.

In one embodiment of the present invention, the subtilase is selected from the following:

-   -   subtilisin from Bacillus amyloliquefaciens BPN′ (described by         Vasantha et al. (1984) J. Bacteriol. Volume 159, p. 811-819 and         JA Wells et al. (1983) in Nucleic Acids Research, Volume 11, p.         7911-7925),     -   subtilisin from Bacillus licheniformis (subtilisin Carlsberg;         disclosed in E L Smith et al. (1968) in J. Biol Chem, Volume         243, pp. 2184-2191, and Jacobs et al. (1985) in Nucl. Acids Res,         Vol 13, p. 8913-8926),     -   subtilisin PB92 (original sequence of the alkaline protease PB92         is described in EP 283075 A2),     -   subtilisin 147 and/or 309 (Esperase®, Savinase®) as disclosed in         GB 1243784,     -   subtilisin from Bacillus lentus as disclosed in WO 91/02792,         such as from Bacillus lentus DSM 5483 or the variants of         Bacillus lentus DSM 5483 as described in WO 95/23221,     -   subtilisin from Bacillus alcalophilus (DSM 11233) disclosed in         DE 10064983,     -   subtilisin from Bacillus gibsonii(DSM 14391) as disclosed in WO         2003/054184,     -   subtilisin from Bacillus sp. (DSM 14390) disclosed in WO         2003/056017,     -   subtilisin from Bacillus sp. (DSM 14392) disclosed in WO         2003/055974,     -   subtilisin from Bacillus gibsonii(DSM 14393) disclosed in WO         2003/054184,     -   subtilisin having SEQ ID NO: 4 as described in WO 2005/063974 or         a subtilisin which is at least 40% identical thereto and having         proteolytic activity,     -   subtilisin having SEQ ID NO: 4 as described in WO 2005/103244 or         subtilisin which is at least 80% identical thereto and having         proteolytic activity,     -   subtilisin having SEQ ID NO: 7 as described in WO 2005/103244 or         subtilisin which is at least 80% identical thereto and having         proteolytic activity, and     -   subtilisin having SEQ ID NO: 2 as described in application DE         102005028295.4 or subtilisin which is this at least 66%         identical thereto and having proteolytic activity.

Examples of useful proteases (C) in accordance with the present invention comprise the variants described in: WO 92/19729, WO 95/23221, WO 96/34946, WO 98/20115, WO 98/20116, WO 99/11768, WO 01/44452, WO 02/088340, WO 03/006602, WO 2004/03186, WO 2004/041979, WO 2007/006305, WO 2011/036263, WO 2011/036264, and WO 2011/072099. Suitable examples comprise especially protease variants of subtilisin protease derived from SEQ ID NO:22 as described in EP 1921147 (which is the sequence of mature alkaline protease from Bacillus lentus DSM 5483) with amino acid substitutions in one or more of the following positions: 3, 4, 9, 15, 24, 27, 33, 36, 57, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 (according to the BPN′ numbering), which have proteolytic activity. In one embodiment, such a subtilisin protease is not mutated at positions Asp32, His64 and Ser221 (according to BPN′ numbering).

In one embodiment, subtilisin has SEQ ID NO:22 as described in EP 1921147, or a subtilisin which is at least 80% identical thereto and has proteolytic activity. In one embodiment, a subtilisin is at least 80% identical to SEQ ID NO:22 as described in EP 1921147 and is characterized by having amino acid glutamic acid (E), or aspartic acid (D), or asparagine (N), or glutamine (Q), or alanine (A), or glycine (G), or serine (S) at position 101 (according to BPN′ numbering) and has proteolytic activity. In one embodiment, subtilisin is at least 80% identical to SEQ ID NO:22 as described in EP 1921147 and is characterized by having amino acid glutamic acid (E), or aspartic acid (D), at position 101 (according to BPN′ numbering) and has proteolytic activity. Such a subtilisin variant may comprise an amino acid substitution at position 101, such as R101E or R101D, alone or in combination with one or more substitutions at positions 3, 4, 9, 15, 24, 27, 33, 36, 57, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and/or 274 (according to BPN′ numbering) and has proteolytic activity.

In another embodiment, a subtilisin is at least 80% identical to SEQ ID NO:22 as described in EP 1921147 and is characterized by comprising at least the following amino acids (according to BPN′ numbering) and has proteolytic activity:

-   -   (a) threonine at position 3 (3T)     -   (b) isoleucine at position 4 (4I)     -   (c) alanine, threonine or arginine at position 63 (63A, 63T, or         63R)     -   (d) aspartic acid or glutamic acid at position 156 (156D or         156E)     -   (e) proline at position 194 (194P)     -   (f) methionine at position 199 (199M)     -   (g) isoleucine at position 205 (205I)     -   (h) aspartic acid, glutamic acid or glycine at position 217         (217D, 217E or 217G),     -   (i) combinations of two or more amino acids according to (a) to         (h).

In another embodiment, a subtilisin is at least 80% identical to SEQ ID NO:22 as described in EP 1921147 and is characterized by comprising one amino acid (according to (a)-(h)) or combinations according to (i) together with the amino acid 101E, 101D, 101N, 101Q, 101A, 101G, or 101S (according to BPN′ numbering) and has proteolytic activity.

In one embodiment, a subtilisin is at least 80% identical to SEQ ID NO:22 as described in EP 1921147 and is characterized by comprising the mutation (according to BPN′ numbering) R101E, or S3T+V4+V205I, or S3T+V4+V199M+V205I+L217D and has proteolytic activity.

In another embodiment, the subtilisin comprises an amino acid sequence having at least 80% identity to SEQ ID NO:22 as described in EP 1921147 and being further characterized by comprising R101E and S3T, V4I, and V205I (according to the BPN′ numbering) and has proteolytic activity.

In another embodiment, a subtilisin comprises an amino acid sequence having at least 80% identical to SEQ ID NO:22 as described in EP 1921147 and being further characterized by comprising R101E, and one or more substitutions selected from the group consisting of S156D, L262E, Q137H, S3T, R45E,D,Q, P55N, T58W,Y,L, Q59D,M,N,T, G61 D,R, S87E, G97S, A98D,E,R, S106A,W, N117E, H120V,D,K,N, S125M, P129D, E136Q, S144W, S161T, S163A,G, Y171 L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T,D and L262N,Q,D (as described in WO 2016/096711 and according to the BPN′ numbering), and has proteolytic activity.

Percentage-identity for subtilisin variants is calculated as disclosed above. Subtilisin variant enzymes as disclosed above which are at least n % identical to the respective parent sequences include variants with n being at least 40 to 100. Depending on the %-identity values applicable as provided above, subtilisin variants in one embodiment have proteolytic activity and are at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical when compared to the full length polypeptide sequence of the parent enzyme.

In another embodiment, the invention relates to subtilisin variants comprising conservative mutations not pertaining the functional domain of the respective subtilisin protease. Depending on the %-identity values applicable as provided above, subtilisin variants of this embodiment have proteolytic activity and are at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar when compared to the full length polypeptide sequence of the parent enzyme.

Proteases (C), including serine proteases, according to the invention have “proteolytic activity” or “protease activity” or “proteolytic activity”. This property is related to hydrolytic activity of a protease (proteolysis, which means hydrolysis of peptide bonds linking amino acids together in a polypeptide chain) on protein containing substrates, e.g. casein, hemoglobin and BSA. Quantitatively, proteolytic activity is related to the rate of degradation of protein by a protease or proteolytic enzyme in a defined course of time. The methods for analyzing proteolytic activity are well-known in the literature (see e.g. Gupta et al. (2002), Appl. Microbiol. Biotechnol. 60: 381-395). Proteolytic activity as such can be determined by using Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA, short AAPF; see e.g. DelMar et al. (1979), Analytical Biochem 99, 316-320) as substrate. pNA is cleaved from the substrate molecule by proteolytic cleavage, resulting in release of yellow color of free pNA which can be quantified by measuring OD₄₀₅.

Protease variants may have proteolytic activity when said protease variants exhibit at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at 10 least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the proteolytic activity of the respective parent protease.

Preferably, the pI value (isoelectric point) of subtilisin protease used in the present invention is in the range of from pH 7.0 to pH 10.0, preferably from pH 8.0 to pH 9.5.

Lipases and Cutinases

In one embodiment, inventive compositions comprise at least one lipase (C). “Lipases”, “lipolytic enzyme”, “lipid esterase”, all refer to an enzyme of EC class 3.1.1 (“carboxylic ester hydrolase”).

Such an enzyme (C) may have lipase activity (or lipolytic activity; triacylglycerol lipase, EC 3.1.1.3), cutinase activity (EC 3.1.1.74; enzymes having cutinase activity may be called cutinase herein), sterol esterase activity (EC 3.1.1.13) and/or wax-ester hydrolase activity (EC 3.1.1.50).

Lipases include those of bacterial or fungal origin.

Commercially available lipase (C) include but are not limited to those sold under the trade names Lipolase™, Lipex™, Lipolex™ and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) and Lipomax (Gist-Brocades/now DSM).

In one aspect of the invention, a suitable lipase is selected from the following: lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258068, EP 305216, WO 92/05249 and WO 2009/109500 or from H. insolens as described in WO 96/13580,

-   -   lipases derived from Rhizomucor mieheias described in WO         92/05249.     -   lipase from strains of Pseudomonas (some of these now renamed to         Burkholderia), e.g. from P. alcalgenes or P. pseudoalcalgenes         (EP 218272, WO 94/25578, WO 95/30744, WO 95/35381, WO         96/00292), P. cepacia(EP 331376), P. stutzeri(GB 1372034), P.         fluorescens, Pseudomonas sp. strain SD705 (WO 95/06720 and WO         96/27002), P. wisconsinensis (WO 96/12012), Pseudomonas         mendocina (WO 95/14783), P. glumae (WO 95/35381, WO 96/00292)     -   lipase from Streptomyces griseus (WO 2011/150157) and S.         pristinaespiralis (WO 2012/137147), GDSL-type Streptomyces         lipases (WO 2010/065455),     -   lipase from Thermobifida fusca as disclosed in WO 2011/084412,     -   lipase from Geobacillus stearothermophilus as disclosed in WO         2011/084417,     -   Bacillus lipases, e.g. as disclosed in WO 00/60063, lipases         from B. subtilis as disclosed in Dartois et al. (1992),         Biochemica et Biophysica Acta, 1131, 253-360 or WO         2011/084599, B. stearothermophilus (JP S64-074992) or B. pumilus         (WO 91/16422).     -   Lipase from Candida antarctica as disclosed in WO 94/01541.     -   cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536, WO         88/09367)     -   cutinase from Magnaporthe grisea (WO 2010/107560),     -   cutinase from Fusarum solani pisi as disclosed in WO 90/09446,         WO 00/34450 and WO 01/92502     -   cutinase from Humicola lanuginosa as disclosed in WO 00/34450         and WO 01/92502

Suitable lipases (C) also include those referred to as acyltransferases or perhydrolases, e.g. acyltransferases with homology to Candida antarctica lipase A (WO 2010/111143), acyltransferase from Mycobacterium smegmatis (WO 2005/056782), perhydrolases from the CE7 family (WO 2009/67279), and variants of the M. smegmatis perhydrolase in particular the S54V variant (WO 2010/100028).

Suitable lipases include also those which are variants of the above described lipases and/or cutinases which have lipolytic activity. Such suitable lipase variants are e.g. those which are developed by methods as disclosed in WO 95/22615, WO 97/04079, WO 97/07202, WO 00/60063, WO 2007/087508, EP 407225 and EP 260105.

Suitable lipases/cutinases (C) include also those that are variants of the above described lipases/cutinases which have lipolytic activity. Suitable lipase/cutinase variants include variants with at least 40 to 100% identity when compared to the full length polypeptide sequence of the parent enzyme as disclosed above. In one embodiment lipase/cutinase variants having lipolytic activity may be at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical when compared to the full length polypeptide sequence of the parent enzyme as disclosed above.

In another embodiment, inventive compositions comprise at least one lipase/cutinase variant comprising conservative mutations not pertaining the functional domain of the respective lipase/cutinase. Lipase/cutinase variants of such embodiments having lipolytic activity may be at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar when compared to the full length polypeptide sequence of the parent enzyme.

Lipases (C) have “lipolytic activity”. The methods for determining lipolytic activity are well-known in the literature (see e.g. Gupta et al. (2003), Biotechnol. Appl. Biochem. 37, p. 63-71). E.g. the lipase activity may be measured by ester bond hydrolysis in the substrate para-nitrophenyl palmitate (pNP-Palmitate, C:16) and releases pNP which is yellow and can be detected at 405 nm.

Lipase variants may have lipolytic activity according to the present invention when said lipase variants exhibit at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at 10 least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the lipolytic activity of the respective parent lipase.

In one embodiment of the present invention, a combination of at least two of the foregoing lipases (C) may be used.

Lipase (C) may be used in its non-purified form or in a purified form, e.g. purified with the aid of well-known adsorption methods, such as phenyl sepharose adsorption techniques.

In one embodiment of the present invention, lipases (C) are included in inventive composition in such an amount that a finished inventive composition has a lipolytic enzyme activity in the range of from 100 to 0.005 LU/mg, preferably 25 to 0.05 LU/mg of the composition. A Lipase Unit (LU) is that amount of lipase which produces 1 μmol of titratable fatty acid per minute in a pH stat. under the following conditions: temperature 30° C.; pH=9.0; substrate is an emulsion of 3.3 wt. % of olive oil and 3.3% gum arabic, in the presence of 13 mmol/I Ca² and 20 mmol/I NaCl in 5 mmol/I Tris-buffer.

In one embodiment of the present invention, proteases (C) are included in inventive composition in such an amount that a finished inventive composition has a proteolytic enzyme activity in the range of from 0.1 to 50 GU.

It is preferred to use a combination of lipase (C) and protease (C) in compositions, for example 1 to 2% by weight of protease (C) and 0.1 to 0.5% by weight of lipase (C).

In the context of the present invention, an enzyme (C) is called stable when its enzymatic activity “available in application” equals 100% when compared to the initial enzymatic activity before storage. An enzyme may be called stable within this invention if its enzymatic activity available in application is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% when compared to the initial enzymatic activity before storage.

In one embodiment, lipolytic activity available after storage at 37° C. for 30 days is at least 60% when compared to the initial lipolytic activity before storage.

Subtracting a % from 100% gives the “loss of enzymatic activity during storage” when compared to the initial enzymatic activity before storage. In one embodiment, an enzyme is stable according to the invention when essentially no loss of enzymatic activity occurs during storage, i.e. loss in enzymatic activity equals 0% when compared to the initial enzymatic activity before storage. Essentially no loss of enzymatic activity within this invention may mean that the loss of enzymatic activity is less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% when compared to the initial enzymatic activity before storage.

In one embodiment, the loss of lipolytic activity after storage at 37° C. for 30 days is less than 40% when compared to the initial lipolytic activity before storage.

Reduced loss of enzymatic activity within this invention may mean that the loss of enzymatic activity is reduced by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% when compared to the initial enzymatic activity before storage.

In one embodiment of the present invention salts (A) are used in combination with serine protease inhibitors known per se. Examples of serine protease inhibitors are boric acid, a borate, or another boronic acid derivative or a peptide aldehyde. The inhibitor may have an inhibition constant to a serine protease Ki (M, mol/L) of 1E-12-1E-03; more preferred 1E-11-1E-04; even more preferred: 1E-10-1E-05; even more preferred 1E-10-1E-06; most preferred 1E-09-1E-07.

It is preferred, though, to use salt (A) in inventive formulations without boron based inhibitors.

Inventive compositions are preferably used as detergent compositions. It is possible, though, to use inventive compositions in further fields, for example in the manufacture of leather. In such an inventive composition, enzyme (C) and preferably lipase (C) may be present in an amount of 0.01 to 4.0% by weight, preferred are 0.1 to 1.5% by weight. In non-detergent applications, a weight ratio of salt (A):lipase (C) in the range of from 0.5:1 to 50:1 is preferred, more preferred from 5:1 to 20:1 and even more preferred 5:1 to 10:1. Other ingredients in a non-detergent liquid compositions may be selected from polyols and mixtures of polyols, for example sorbitol, glycerol, and propylene glycol, each in amounts of 1 to 60%, small amounts of non-ionic surfactants (ex softanol) 0-5%, calcium ions 0.001%-0.5%, water. Further examples are preservatives, e.g., 2-phenoxyethanol.

Inventive compositions may contain one or more ingredients other than salt (A), surfactant (B) or enzyme (C), for example organic solvents, fragrances, dyestuffs, biocides, preservatives, hydrotropes, builders, viscosity modifiers, polymers, buffers, defoamers, and anti-corrosion additives.

In one embodiment of the present invention, compositions according to the present invention may comprise one or more organic solvents, for example ethanol, n-propanol, iso-propanol, n-butanol, iso-butanol, sec.-butanol, ethylene glycol, propylene glycol, 1,3-propane diol, butane diol, glycerol, diglycol, propyl diglycol, butyl diglycol, hexylene glycol, ethylene glycol methyl ether, ethylene glycol ethyl ether, ethylene glycol propyl ether, and phenoxyethanol, preferred are ethanol, isopropanol or propylene glycol. Preferred amounts of organic solvents are 0.5 to 25% by weight, referring to the entire inventive composition. Especially when inventive compositions are delivered in pouches or the like, 8 to 25% by weight of organic solvent(s).

Examples of fragrances are benzyl salicylate, 2-(4-tert.-butylphenyl) 2-methylpropional, commercially available as Lilial®, and hexyl cinnamaldehyde.

Examples of dyestuffs are Acid Blue 9, Acid Yellow 3, Acid Yellow 23, Acid Yellow 73, Pigment Yellow 101, Acid Green 1, Solvent Green 7, and Acid Green 25.

Inventive liquid detergent compositions may contain one or more preservatives or biocides. Biocides and preservatives prevent alterations of inventive liquid detergent compositions due to attacks from microorganisms. Examples of biocides and preservatives are BTA (1,2,3-benzotriazole), benzalkonium chlorides, 1,2-benzisothiazolin-3-one (“BIT”), 2-methyl-2H-isothiazol-3-one (“MIT”) and 5-chloro-2-methyl-2H-isothiazol-3-one (“CIT”), benzoic acid, sorbic acid, iodopropynyl butylcarbamate (“IPBC”), dichlorodimethylhydantoine (“DCDMH”), bromochlorodimethylhydantoine (“BCDMH”), and dibromodimethylhydantoine (“DBDMH”).

Examples of viscosity modifiers are agar-agar, carragene, tragacanth, gum arabic, alginates, pectins, hydroxyethyl cellulose, hydroxypropyl cellulose, starch, gelatin, locust bean gum, cross-linked poly(meth)acrlyates, for example polyacrlyic acid cross-linked with bis-(meth)acrylamide, furthermore silicic acid, clay such as—but not limited to—montmorrilionite, zeolite, dextrin, and casein.

Hydrotropes in the context with the present invention are compounds that facilitate the dissolution of compounds that exhibit limited solubilty in water. Examples of hydrotropes are organic solvents such as ethanol, isopropanol, ethylene glycol, 1,2-propylene glycol, and further organic solvents that are water-miscible under normal conditions without limitation. Further examples of suitable hydrotropes are the sodium salts of toluene sulfonic acid, of xylene sulfonic acid, and of cumene sulfonic acid.

Examples of polymers are especially polyacrylic acid and its respective alkali metal salts, especially its sodium salt. A suitable polymer is in particular polyacrylic acid, preferably with an average molecular weight M_(w) in the range from 2,000 to 40,000 g/mol. preferably 2,000 to 10,000 g/mol, in particular 3,000 to 8,000 g/mol, each partially or fully neutralized with alkali, especially with sodium. Also of suitability are copolymeric polycarboxylates, in particular those of acrylic acid with methacrylic acid and of acrylic acid or methacrylic acid with maleic acid and/or fumaric acid. Polyacrylic acid and its respective alkali metal salts may serve as soil anti-redeposition agents.

Further examples of polymers are polyvinylpyrrolidones (PVP). Polyvinylpyrrolidones may serve as dye transfer inhibitors.

Further examples of polymers are polyethylene terephthalates, polyoxyethylene terphthalates, and polyethylene terephthalates that are end-capped with one or two hydrophilic groups per molecule, hydrophilic groups being selected from CH₂CH₂CH₂—SO₃Na, CH₂CH(CH₂—SO₃Na)₂, and CH₂CH(CH₂SO₂Na)CH₂—SO₃Na.

Examples of buffers are monoethanolamine and N,N,N-triethanolamine.

Examples of defoamers are silicones.

In one embodiment of the present invention, inventive compositions may contain one or more builders other than salt (A).

Inventive compositions may contain 1 to 40% by weight of a detergent builder other than salt (A), such as, but not limited to zeolite, phosphate, phosphonate, citrate, polymer builders, or aminocarboxylates such as the alkali metal salts of iminodisuccinates, for example IDS-Na₄, furthermore nitrilotriacetic acid (“NTA”), methylglycine diacetic acid (“MGDA”), glutamic acid diacetic acid (“GLDA”), ethylene diamine tetraacetic acid (“EDTA”) or diethylenetriamine pentaacetic acid (“DTPA”). Preferred alkali metal salts are the potassium salts and especially the sodium salts.

Further examples of detergent builders are polymers with complexing groups like, for example, polyethylenimine in which 20 to 90 mole-% of the N-atoms bear at least one CH₂COO⁻ group, and the respective alkali metal salts of the above sequestrants, especially their sodium salts.

Further examples of suitable polymers are polyalkylenimines, for example polyethylenimines and polypropylene imines. Polyalkylenimines may be used as such or as polyalkoxylated derivatives, for examples ethoxylated or propoxylated. Polyalkylenimines contain at least three alkylenimine units per molecule.

In one embodiment of the present invention, said alkylenimine unit is a C₂-C₁₀-alkylendiamine unit, for example a 1,2-propylendiamine, preferably an α,ω-C₂-C₁₀-alkylendiamine, for example 1,2-ethylendiamine, 1,3-propylendiamine, 1,4-butylendiamine, 1,5-pentylendiaminne, 1,6-hexandiamine (also being referred to as 1,6-hexylendiamine), 1,8-diamine or 1,10-decandiamine, even more preferred are 1,2-ethylendiamine, 1,3-propylendiamine, 1,4-butylendiamine, and 1,6-hexandiamine.

In another embodiment of the present invention, said polyalkylenimine is selected from polyalkylenimine unit, preferably a polyethylenimine or polypropylenimine unit.

The term “polyethylenimine” in the context of the present invention does not only refer to polyethylenimine homopolymers but also to polyalkylenimines containing NH—CH₂—CH₂—NH structural elements together with other alkylene diamine structural elements, for example NH—CH₂—CH₂—CH₂—NH structural elements, NH—CH₂—CH(CH₃)—NH structural elements, NH—(CH₂)₄—NH structural elements, NH—(CH₂)—NH structural elements or (NH—(CH₂)—NH structural elements but the NH—CH₂—CH₂—NH structural elements being in the majority with respect to the molar share. Preferred polyethylenimines contain NH—CH₂—CH₂—NH structural elements being in the majority with respect to the molar share, for example amounting to 60 mol-% or more, more preferably amounting to at least 70 mol-%, referring to all alkylenimine structural elements. In a special embodiment, the term polyethylenimine refers to those polyalkylenimines that bear only one or zero alkylenimine structural element per polyethylenimine unit that is different from NH—CH₂—CH₂—NH.

The term “polypropylenimine” in the context of the present invention does not only refer to polypropylenimine homopolymers but also to polyalkylenimines containing NH—CH₂—CH(CH₃)—NH structural elements together with other alkylene diamine structural elements, for example NH—CH₂—CH₂—CH₂—NH structural elements, NH—CH₂—CH₂—NH structural elements, NH—(CH₂)₄—NH structural elements, NH—(CH₂)₆—NH structural elements or (NH—(CH₂)₈—NH structural elements but the NH—CH₂—CH(CH₃)—NH structural elements being in the majority with respect to the molar share. Preferred polypropylenimines contain NH—CH₂—CH(CH₃)—NH structural elements being in the majority with respect to the molar share, for example amounting to 60 mol-% or more, more preferably amounting to at least 70 mol-%, referring to all alkylenimine structural elements. In a special embodiment, the term polypropylenimine refers to those polyalkylenimines that bear only one or zero alkylenimine structural element per polypropylenimine unit that is different from NH—CH₂—CH(CH₃)—NH.

Branches may be alkylenamino groups such as, but not limited to —CH₂—CH₂—NH₂ groups or (CH₂)—NH₂-groups. Longer branches may be, for examples, —(CH₂)₃—N(CH₂CH₂CH₂NH₂)₂ or —(CH₂)₂—N(CH₂CH₂NH₂)₂ groups. Highly branched polyethylenimines are, e.g., polyethylenimine dendrimers or related molecules with a degree of branching in the range from 0.25 to 0.95, preferably in the range from 0.30 to 0.80 and particularly preferably at least 0.5. The degree of branching can be determined for example by ¹³C-NMR or ¹⁵N-NMR spectroscopy, preferably in D₂O, and is defined as follows:

DB=D+T/D+T+L

with D (dendritic) corresponding to the fraction of tertiary amino groups, L (linear) corresponding to the fraction of secondary amino groups and T (terminal) corresponding to the fraction of primary amino groups.

Within the context of the present invention, branched polyethylenimine units are polyethylenimine units with DB in the range from 0.25 to 0.95, particularly preferably in the range from 0.30 to 0.90% and very particularly preferably at least 0.5. Preferred polyethylenimine units are those that exhibit little or no branching, thus predominantly linear or linear polyethylenimine units.

In the context of the present invention, CH₃-groups are not being considered as branches.

In one embodiment of the present invention polyalkylenimine may have a primary amine value in the range of from 1 to 1000 mg KOH/g, preferably from 10 to 500 mg KOH/g, most preferred from 50 to 300 mg KOH/g. The primary amine value can be determined according to ASTM D2074-07.

In one embodiment of the present invention polyalkylenimine may have a secondary amine value in the range of from 10 to 1000 mg KOH/g, preferably from 50 to 500 mg KOH/g, most preferred from 50 to 500 mg KOH/g. The secondary amine value can be determined according to ASTM D2074-07.

In one embodiment of the present invention polyalkylenimine may have a tertiary amine value in the range of from 1 to 300 mg KOH/g, preferably from 5 to 200 mg KOH/g, most preferred from 10 to 100 mg KOH/g. The tertiary amine value can be determined according to ASTM D2074-07.

In one embodiment of the present invention, the molar share of tertiary N atoms is determined by ¹⁵N-NMR spectroscopy. In cases that tertiary amine value and result according to ¹³C-NMR spectroscopy are inconsistent, the results obtained by ¹³C-NMR spectroscopy will be given preference.

In one embodiment of the present invention, the average molecular weight M_(w) of said polyalkylenimine is in the range of from 250 to 100,000 g/mol, preferably up to 50,000 g/mol and more preferably from 800 up to 25,000 g/mol. The average molecular weight M_(w) of polyalkylenimine may be determined by gel permeation chromatography (GPC) of the intermediate respective polyalkylenimine, with 1.5% by weight aqueous formic acid as eluent and cross-linked polyhydroxyethyl methacrylate as stationary phase.

Said polyalkylenimine may be free or alkoxylated, said alkoxylation being selected from ethoxylation, propoxylation, butoxylation and combinations of at least two of the foregoing. Preference is given to ethylene oxide, 1,2-propylene oxide and mixtures of ethylene oxide and 1,2-propylene oxide. If mixtures of at least two alkylene oxides are applied, they can be reacted step-wise or simultaneously.

In one embodiment of the present invention, an alkoxylated polyalkylenimine bears at least 6 nitrogen atoms per unit.

In one embodiment of the present invention, polyalkylenimine is alkoxylated with 2 to 50 moles of alkylene oxide per NH group, preferably 5 to 30 moles of alkylene oxide per NH group, even more preferred 5 to 25 moles of ethylene oxide or 1,2-propylene oxide or combinations therefrom per NH group. In the context of the present invention, an NH₂ unit is counted as two NH groups. Preferably, all—or almost all—NH groups are alkoxylated, and there are no detectable amounts of NH groups left.

Depending on the manufacture of such alkoxylated polyalkylenimine, the molecular weight distribution may be narrow or broad. For example, the polydispersity Q=M_(w)/M_(n) in the range of from 1 to 3, preferably at least 1.2 and more preferably 1.2 to 2.5, or it may as well be greater than 3 and up to 20, for example 3.5 to 15 and—in case of broad molecular weight distributions—more preferred in the range of from 4 to 5.5.

In one embodiment of the present invention, the polydispersity Q of alkoxylated polyalkylenimine is in the range of from 2 to 10.

In one embodiment of the present invention alkoxylated polyalkylenimine is selected from polyethoxylated polyethylenimine, ethoxylated polypropylenimine, ethoxylated α,ω-hexandiamines, ethoxylated and propoxylated polyethylenimine, ethoxylated and propoxylated polypropylenimine, and ethoxylated and poly-propoxylated α,ω-hexandiamines.

In one embodiment of the present invention the average molecular weight Mn (number average) of alkoxylated polyethylenimine is in the range of from 2,500 to 1,500,000 g/mol, determined by GPC, preferably up to 500,000 g/mol.

In one embodiment of the present invention, the average alkoxylated polyalkylenimine are selected from ethoxylated α,ω-hexanediamines and ethoxylated and poly-propoxylated α,ω-hexanediamines, each with an average molecular weight Mn (number average) in the range of from 800 to 50,000 g/mol.

In an alternative embodiment of the present invention, inventive compositions are unbuilt, i.e. essentially free of additional detergent builder.

In one embodiment of the present invention, inventive liquid compositions may comprise from about 0.1% to about 15%, more particularly from 0.25% to 10%, and most particularly from about 0.5% to 5% of salt (A).

Thus, a stabilized liquid enzyme formulation may contain 0.5 to 20% by weight, particularly 1-10% by weight, of enzyme (C) (total of protease and optional second enzyme) and 0.01% to 10% of salt (A), more particularly 0.05 to 5% by weight and most particularly 0.1% to 2% by weight of salt (A).

Succinate builders are preferably used in the form of their water-soluble salts, including the sodium, potassium. ammonium and alkanolammonium salts. Specific examples of succinate builders include: laurylsuccinate, myristylsuccinate, palmitylsuccinate, 2-dodecenylsuccinat (preferred), 2-pentadecenylsuccinate, and the like. Laurylsuccinates are the preferred builders of the above group.

Inventive compositions allow a flexible formulation, even with high or low water content. It was further the objective to provide a composition that has excellent shelf life, especially with respect to the enzyme(s) contained therein. Inventive compositions are therefore excellently suited for laundering of fabrics. Therefore, a further aspect of the present invention is directed towards the use of inventive compositions for the laundering of fabrics. In particular, liquid inventive formulations are excellently suited for laundering of fabrics.

Inventive detergent compositions may be used as fabric cleaning compositions, hard surface cleansing compositions, light duty cleaning compositions including dish cleansing compositions and automatic dishwasher detergent compositions.

A further aspect of the present invention is directed towards the use of a salt (A) for the stabilization of enzymes. Salts (A) have been defined in more details above.

A further aspect of the present invention is a process for making inventive compositions, hereinafter also referred to as inventive manufacturing process. The inventive manufacturing process comprises the steps of

-   (a) mixing     -   (A) at least one salt (A), and     -   (C) at least one enzyme, -   (b) adding at least one surfactant (B) selected from amphoteric and     anionic and non-ionic surfactants.

Steps (a) and (b) may be performed in any order. However, in embodiments wherein surfactant (B) may lead to foam formation it is preferred to first mix salt (A) and enzyme (C) and to then add surfactant (B).

Mixing of salt (A) and enzyme (C) may be performed in any type of vessel. Preferably, foaming and high shear rates during mixing are avoided. Preferred devices are tubular mixers and static mixers.

In a preferred embodiment of the inventive manufacturing process, salt (A) is a compound of general formula (I)

(R²)₃N⁺—(CH₂)_(n)C(R³)(R⁴)—(O—X)_(m)O—C(O)—R¹ A⁻  (I)

wherein

n is selected from 1 to 12,

m is selected from zero to 50,

R¹ is selected from C₁-C₁₀-alkyl, linear or branched, and C₆-C₁₀-aryl, wherein R¹ may bear one or more hydroxyl or C═O or COOH groups, partially or fully neutralized, if applicable,

R² are same or different and selected from C₁-C₁₀-alkyl, phenyl,

R³ and R⁴ are same or different and selected from hydrogen and C₁-C₄-alkyl,

X is C₂-C₄-alkylen, and

A⁻ is a counteranion, inorganic or organic. The variables R¹, R², m, n and A⁻ are as defined above. Preferably, R² in compound according to general formula (I) are all methyl.

In a preferred embodiment of the inventive manufacturing process, salt (A) has a counterion selected from halide, sulphate, carbonate, tartrate, citrate, lactate, and methanesulfonate.

In a preferred embodiment of the inventive manufacturing process the at least on enzyme (C) is selected from protease, amylase, and lipase.

The invention will be further illustrated by working examples.

General remarks: percentages are weight percent unless specifically noted otherwise.

Weight % of enzymes refer to the enzyme preparation as used and available commercially—the weight % of pure protein is quite lower—hence the turn over number/mg as described is important.

Acetylcholine (A.12) was purchased from Sigma Aldrich. The counterion was chloride.

The precursor of (A.14) can be produced directly instead of use of HCl in the ethoxylation of trimethylamine or via reaction of choline hydrogencarbonate with methanesulfonic, see Constantinescu et al in Chem. Eng. Data, 2007, 521280-1285.

I. Synthesis of Salts (A)

Based upon the amounts of water distilled off and by IR spectroscopy it could be shown that the esterification reactions were complete.

90% methanesulfonic acid refers to a mixture from 10% water and 90% methanesulfonic acid.

I.1 Synthesis of Inventive Salt (A.1)

An amount of 225 g (1.5 mole) tartaric acid was dissolved in 280 g of a 75% by weight aqueous solution of choline chloride (1.5 mole). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)—oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 15 g of 90% by weight aqueous methanesulfonic acid were added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 200 g diethylene glycol. An amount of 617 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 7.8 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.1) was obtained.

I.2 Synthesis of Inventive Salt (A.2) An amount of 150 g tartaric acid (1.0 mole) was dissolved in 374 g of a 75% by weight aqueous solution of choline chloride (2.0 mole). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)—oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 15 g of 90% by weight methanesulfonic acid were added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 200 g diethylene glycol. An amount of 607 g of a yellowish liquid were obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 8.7 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.2) was obtained.

I.3 Synthesis of Inventive Salt (A.3)

An amount of 210 g citric acid monohydrate (1.0 mole) was dissolved in 374 g of a 75% by weight aqueous solution of choline chloride (2.0 moles). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)—oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 18 g of 90% by weight methanesulfonic acid were added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation, the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 200 g diethylene glycol. An amount of 607 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 10.3 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.3) was obtained.

I.4 Synthesis of Inventive Salt (A.4)

An amount of 210 g citric acid monohydrate (1.0 mol) was dissolved in 561 g of a 75% by weight aqueous solution of choline chloride (3.0 moles). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)—oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 18 g of 90% by weight methanesulfonic acid were added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 270 g diethylene glycol. An amount of 868 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 9.6 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.4) was obtained.

I.5 Synthesis of Inventive Salt (A.5)

An amount of 210 g citric acid monohydrate (1.0 mol) were dissolved in 485 g of a 75% by weight aqueous solution of choline methanesulfonate (2.0 mol). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)—oil bath temperature of 100 to 120° C., 50 to 80 mbar.

An amount of 18 g of 90% by weight methanesulfonic acid were added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 200 g diethylene glycol. An amount of 663 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 12.5 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.5) was obtained.

I.6 Synthesis of Inventive Salt (A.6)

An amount of 98.1 g maleic anhydride (1.0 mol) were mixed with 363 g of choline methanesulfonate (2.0 moles) as dry substance. The mixture was heated in a rotary evaporator to 135° C.

After one hour of mixing an amount of 12 g of methanesulfonic acid (pure) was added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of mixing the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 200 g diethylene glycol. An amount of 653 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 8.9 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.6) was obtained.

I.7 Synthesis of Inventive Salt (A.7)

An amount of 210 g citric acid monohydrate (1.0 mole) was dissolved in 437 g of a 70% by weight aqueous solution of beta-methyl choline chloride (HO—CH(CH₃—CH₂—N(CH₃)₃ Cl, 2.0 moles). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)—oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 18 g of 90% by weight methanesulfonic acid were added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 200 g diethylene glycol. An amount of 676 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 12.3 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.7) was obtained.

I.8 Synthesis of Inventive Salt (A.8)

An amount of 105 g citric acid monohydrate (0.5 moles) was dissolved in 327 g of a 70% by weight aqueous solution of beta-methyl choline chloride (1.5 moles). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)—oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 13 g of 90% methanesulfonic acid by weight methanesulfonic acid was added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 170 g diethylene glycol. An amount of 471 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 21.7 g triethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.8) was obtained.

I.9 Synthesis of Inventive Salt (A.9)

An amount of 210 g citric acid monohydrate (1.0 mole) was dissolved in 520 g of a 70% by weight aqueous solution of beta-n-propyl choline chloride (2.0 moles). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)—oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 18 g of 90% methanesulfonic acid was added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 250 g propylene glycol. An amount of 774 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 11.9 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.9) was obtained.

I.10 Synthesis of Inventive Salt (A.10)

An amount of 210 g citric acid monohydrate (1.0 mole) was dissolved in 520 g of a 70% aqueous solution of dimethylmonobutylcholine chloride (2.0 moles). Water was removed within 45 minutes in a rotary evaporator (2-l-flask)—oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 18 g of 90% methanesulfonic acid was added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 200 g propylene glycol. An amount of 788 g of a yellowish liquid. An aliquot of 200 g of the liquid so obtained was neutralized with 11.4 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.10) was obtained.

I.11 Synthesis of Inventive Salt (A.11)

An amount of 105 g citric acid monohydrate (0.5 moles) was dissolved in 397 g of a 60% by weight aqueous solution of dimethyl n-octylcholine chloride (1.0 mole). Water was removed within 90 minutes in a rotary evaporator (2-l-flask)—oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 9.5 g of 90% methanesulfonic acid was added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 200 g propylene glycol. An amount of 470 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 8.9 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.11) was obtained.

I.13 Synthesis of Inventive Salt (A.13)

An amount of 85 g gallic acid (3,4,5-trihydroxybenzoic-acid, 0.5 moles) was dispersed in 121 g of a 75% by weight aqueous solution of choline methanesulfonate (0.5 moles). Water was removed within 90 minutes in a rotary evaporator (2-l-flask)—oil bath temperature of 100 to 120° C., 50 to 80 mbar. An amount of 8 g of 90% methanesulfonic acid was added and the temperature was raised to 145° C. at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145° C. A light yellowish substance was obtained that was diluted with 100 g diethylene glycol. An amount of 271 g of a yellowish liquid was obtained. An aliquot of 100 g of the liquid so obtained was neutralized with 4.6 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.13) was obtained.

Comparative Salts:

C-(A.15) Choline chloride, 75% by weight aqueous solution, commercially available from BASF SE

C-(A.16) An amount of 75 g (0.5 mol) tartaric acid was portion-wise dissolved (15 g units) in 206 g of an 80% by weight aqueous solution of choline bicarbonate (1.0 mol). The solution was stirred until the CO₂ evolution ceased. Water was removed within 90 minutes by rotary evaporation (2-l-flask)—oil bath temperature of 120° C., 10 mbar. A clear substance was obtained that was diluted with 150 g diethylene glycol. 390 g of a clear solution were obtained, C-(A.16). No ester formation could be detected.

C-(A.17): An amount of 105 g (0.5 mol) citric acid monohydrate was portion-wise dissolved (20 g units) in 206 g of an 80% by weight aqueous solution of choline bicarbonate (1.0 mol). The solution was stirred until the CO₂ evolution ceased. Water was removed within 90 minutes by rotary evaporation (2-l-flask)—oil bath temperature of 120° C., 10 mbar. A clear substance was obtained that was diluted with 150 g diethylene glycol. 412 g of a clear solution were obtained, C-(A.17). No ester formation could be detected.

C-(A.18): An amount of 105 g (0.5 mol) citric acid monohydrate was portion-wise dissolved (20 g units) in 309 g of an 80% by weight aqueous solution of choline bicarbonate (1.5 moles). The solution was stirred until the CO₂ evolution ceased. Water was removed within 90 minutes by rotary evaporation (2-l-flask)—oil bath temperature of 120° C., 10 mbar. A clear substance was obtained that was diluted with 150 g diethylene glycol. 497 g of a clear viscous solution were obtained, C-(A.18). No ester formation could be detected.

C-(A.19): citric acid monohydrate, C-(A.20): monosodium salt of citric acid, C-(A.21): disodium salt of citric acid, C-(A.22): trisodium salt of citric acid. C-(A.19), C-(A.20), C-(A.21) and C-(A.22) are known builder compounds used in detergents formulations.

II. Application Tests

II.1 Formation of Stable Liquid Formulations

Low water liquid detergent formulations substitute water with glycols as like diethylene glycol or DPG and hence solubility is inevitable. Salts (A.1) to (A.11) and C-(A.16) to C-(A.18) are each soluble in diethylene glycol and/or dipropylene glycol and hence can be formulated without water.

50 g of C-(A.19), C-(A.20), C-(A.21) and C-(A.22) were each combined in a flask together with 100 g diethylene glycol and heated at 100° C. for 30 minutes under stirring. The heating source was removed and the resulting white suspensions were cooled to ambient temperature over a period of 10 hours. The resulting slurries were filtered (paper filter) and the filter cakes washed twice with 50 g isopropanol. The isolated compounds C-(A.19), C-(A.20), C-(A.21) and C-(A.22) were gravimetrically determined, showing that in the absence of water neither C-(A.19) nor C-(A.20) nor C-(A.21) nor C-(A.22) cannot be used due to insufficient solubility. The following amounts were obtained as filter cakes:

C-(A.19): 44.0 g; C-(A.20): 45.8 g; C-(A.21): 47.2 g; C-(A.22): 48.2 g

II.2 Enzyme Stability Tests

The storage stability of lipase and protease in water was assessed at 37° C.

Base test formulations were manufactured by making base formulations I to VI by mixing the components according to Table 1.

The respective salt (A) or comparative compound was added, if applicable, to the respective base formulation in amounts as indicated in Table 1.

Enzyme (C) was added, to the respective base formulation in amounts as indicated in Table 1. The amount of enzyme as provided in Table 1 refers to active protein. Either lipase or protease was added, depending on which enzyme activity was measured.

Lipolase® 100 L (CAS-No. 9001-62-1, EC-No. 232-619-9) was purchased from Sigma-Aldrich. Savinase® 16.0 L (CAS-No. 9014-01-1, EC-No. 232-752-2) was purchased from Sigma-Aldrich.

Water was added to accomplish the balance to 100.

Lipolase activity at certain points in time as indicated in Table 2 was be determined by employing pNitrophenol-valerate (2.4 mM pNP-C5 in 100 mM Tris pH 8.0, 0.01% Triton X100) as a substrate. The absorption was measured at 20° C. every 30 seconds over 5 minutes at 405 nm.

The slope (absorbance increase at 405 nm per minute) of the time dependent absorption-curve is directly proportional to the activity of the lipase.

Table 2 shows lipase activity in liquid formulations measured after storage; 1-30 days at 37° C. The lipolytic activity values provided in Table 2 were calculated referring to the 100% value determined in the reference formulation at the time 0.

The nomenclature of formulations is as follows: the Roman number before the full stop characterizes the base formulation, the Arabian number the type of salt. Zero: no salt (A). “C—”: comparative formulations.

TABLE 1 liquid laundry formulations wt % in formulation Ingredients I II III IV V VI Reference (B.1) 15 8 — 35 30 25 6 (B.2) — 6 8 — — — — (B.3) 6 4 — 8 — 22 7.5 (B.4) 2 — — 10 12 6 2 (B.5) 4 8 4 14 — 8 (B.6) — 2.5 — — 5 — — Sorbitol 3 — — 3 — — — PEI-EO20 3 5 3 5 5 — — Propyleneglycol 4 — 8 6 4 8 Glycerol — — 6 — 6 8 Ethanol — — — — — — 2.5 Ca-formiate 1 — 1 2 2 — Savinase 0.5 0.5 0.5 0.5 0.5 0.5 Lipolase* 0.4 0.4 0.4 0.4 0.4 0.4 (A) 2.5 2.5 2.5 4.0 4.0 4.0 balance Water to 100 (B.1): n-C₁₈-alkyl-(OCH₂CH₂)₂₅—OH (B.2): C₁₀-C₁₈-alkylpolygycoside blend (B.3): Sodium C₁₀-C₁₂-alkyl benzenesulfonate (B.4): Sodium cumenesulfonate (B.5): Sodium laurethsulfate —n-C₁₂H₂₅—O—(CH₂CH₂O)₃—SO₃Na (B.6): n-C₁₂H₂₅(CH₃)₂N→O *for comparative tests the experiments were repeated without enzymes * **for comparative tests without salts (A) said amounts of salt (A) were replaced with the same amount of diethylene glycol.

The enzyme activity of the protease was measured in a Thermo Fisher Scientific Gallery one-channel interference filter photometer (made by Thermo Fisher Scientific) by titration with N-succinyl-Ala-Ala-Pro-Phe-p-pitroanilide in DMSO. The release of para-nitroaniline resulted in an increase of absorbance at 405 nm and is proportional to the enzyme activity measured in PROT units. One PROT is the amount of enzyme that releases 1 μmol of para-nitroaniline from I mM of N-succinyl-Ala-Ala-Pro-Phe-p-pitroanilide per minute at pH of 9.0 and 37° C. In Table 2, lipase activity in formulations and comparative formulations was determined after storage; 1-30 days at 37° C. An activity of 100% refers to the activity in aqueous environment and identical concentration.

TABLE 2 results of stability tests Base formul. (A) T0 1 d 3 d 6 d 10 d 15 d 20 d 25 d 30 d C-I.0 nil 95 89 77 68 53 38 30 22 16 I.1 (A.1) 96 96 93 94 89 85 82 64 72 I.3 (A.3) 101 100 98 95 91 88 85 68 78 I.4 (A.4) 103 100 99 96 95 93 90 86 85 I.6 (A.6) 97 96 94 92 89 85 80 78 75 I.7 (A.7) 95 95 91 85 81 76 69 65 59 C-I.15 C-(A.15) 97 95 80 67 55 41 32 22 20 C-I.16 C-(A.16) 95 90 81 68 51 40 34 25 24 C-I.17 C-(A.17) 97 90 80 70 53 42 38 33 29 C-I.18 C-(A.18) 98 91 84 72 55 45 39 32 29 C-II.0 nil 94 92 81 73 57 41 32 25 20 II.2 (A.2) 95 94 92 90 88 84 80 75 68 II.5 (A.5) 102 100 97 95 93 89 86 72 79 II.6 (A.6) 103 100 99 96 94 92 90 86 88 II.8 (A.8) 96 94 90 85 80 80 76 72 68 II.9 (A.9) 97 93 90 87 83 81 77 75 76 II.10 (A.10) 96 96 92 89 83 84 79 76 73 II.12 (A.12) 100 98 96 95 88 86 80 77 76 C-II.15 C-(A.15) 96 95 82 65 56 40 33 26 22 C-II.22 C-(A.22) 95 87 79 67 55 40 33 24 18 C-III.0 nil 96 93 83 74 63 51 42 30 24 III.4 (A.4) 100 98 96 93 90 85 84 82 77 III.6 (A.6) 104 100 101 97 94 90 89 86 82 III.9 (A.9) 97 95 93 88 84 80 77 73 71 III.10 (A.10) 96 96 91 86 82 79 76 73 69 III.11 (A.11) 97 96 93 84 83 76 71 70 63 III.12 (A.12) 102 98 97 95 91 86 80 78 74 C- C-(A.20) 100 92 79 70 51 39 30 22 16 III.20 C- C-(A.21) 101 93 78 68 50 39 31 25 20 III.21 C- C-(A.22) 98 92 76 66 48 37 28 23 19 III.22 C-IV.0 nil 88 85 81 70 60 55 46 39 33 IV.1 (A.1) 98 96 93 92 87 84 82 79 71 IV.3 (A.3) 99 100 98 95 90 88 85 80 74 IV.4 (A.4) 101 100 97 93 90 89 86 81 73 IV.6 (A.6) 96 96 91 89 86 85 81 78 75 IV.7 (A.7) 97 95 90 85 81 78 73 70 64 C- C-(A.15) 94 95 82 72 59 44 36 30 23 IV.15 C- C-(A.16) 95 90 81 67 55 41 34 28 25 IV.16 C- C-(A.17) 96 93 86 74 63 51 46 38 33 IV.17 C- C-(A.18) 95 91 84 72 58 49 46 39 35 IV.18 C-V.0 nil 83 80 75 68 59 50 41 36 30 V.2 (A.2) 97 94 90 87 84 81 78 74 69 V.4 (A.4) 101 98 94 90 87 84 80 76 71 V.5 (A.5) 101 100 98 96 94 88 84 77 74 V.7 (A.7) 96 95 92 88 84 80 75 70 66 V.11 (A.11) 97 96 91 89 85 77 73 68 60 V.13 (A.13) 95 96 91 87 80 70 61 52 45 C- C-(A.15) 98 95 86 74 63 54 41 39 30 V.15 C- C-(A.16) 96 92 85 70 65 58 49 37 28 V.16 C-VI.0 nil 82 79 72 63 54 47 38 30 25 VI.4 (A.4) 102 99 96 91 86 82 80 75 65 VI.5 (A.5) 99 97 95 91 83 78 73 70 63 VI.6 (A.6) 96 90 87 84 81 74 70 67 61 VI.11 (A.11) 97 92 88 84 83 78 75 72 65 C- C-(A.18) 96 90 83 75 58 50 48 37 32 VI.18 Protease activity: Savinase activity at certain points in time as indicated in Table 3 was be determined by employing Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA, short AAPF) as substrate. pNA is cleaved from the substrate molecule by proteolytic cleavage, resulting in release of yellow color of free pNA which was determined by measuring OD₄₀₅. Measurement were done at 20° C. Table 3 displays protease activity measured in liquid formulations after storage for 1 to 30 days at 37° C. The proteolytic activity values provided in Table 3 were calculated referring to the 100% value determined in the reference formulation at the time 0. The nomenclature of formulations is as follows: the Roman number before the full stop characterizes the base formulation, the Arabian number the type of salt (A.# inventive salt (component (a)); C-(A.#) comparative compound). Zero (“0”): no salt, but diethylene glycol.

TABLE 3 Protease activities with and without salts (A) Base form. (A) T0 1 d 3 d 6 d 10 d 15 d 20 d 25 d 30 d C-I.0 nil 98 98 86 67 49 38 30 23 8 I.1 (A.1) 96 94 91 75 59 48 42 36 29 I.4 (A.4) 100 97 95 76 60 50 45 36 32 I.5 (A.5) 99 96 94 73 58 49 44 38 33 I.6 (A.6) 96 93 87 75 59 50 45 40 29 C-I.15 C-(A.15) 98 92 85 64 47 39 31 22 8 C-I.16 C-(A.16) 98 91 84 66 49 38 30 23 10 C-I.17 C-(A.17) 98 90 81 70 49 38 30 23 12 C-III.0 nil 96 95 86 71 51 40 33 21 12 III.6 (A.6) 92 96 92 82 69 59 50 41 34 III.8 (A.8) 94 95 93 78 68 60 52 42 33 III.12 (A.12) 96 96 92 80 70 61 50 39 31 C- C-(A.17) 92 94 83 71 53 42 34 27 14 III.17 C- C-(A.18) 94 94 85 72 54 43 35 28 14 III.18 C-V.0 nil 83 98 86 70 49 38 30 23 13 V.1 (A.1) 84 93 88 80 62 54 46 38 30 V.2 (A.2) 87 90 90 83 66 58 50 44 36 V.6 (A.6) 88 91 89 84 70 60 52 43 35 C- C-(A.18) 83 90 83 64 50 40 33 25 16 V.18 C-VI.0 nil 87 93 86 70 49 38 30 23 11 VI.4 (A.4) 84 90 88 76 68 61 53 44 30 VI.7 (A.7) 85 90 84 74 66 59 50 40 31 VI.9 (A.9) 83 87 85 75 67 60 51 40 30 VI.11 (A.11) 86 91 87 77 69 58 49 42 32 C- C-(A.15) 85 90 84 66 47 36 29 20 10 VI.15

II.3 Textile Cleaning Tests

The detergent performance of these formulations in cleaning two types of test fabrics was carried out. Testing cloth samples comprised a complex soil containing proteinaceous and fatty components due to CFT process as well as test cloth samples contained a fatty/particulate type of soil.

The test was performed as follows: a multi stain monitor containing 8 standardized soiled fabric patches, each of 2.5×2.5 cm size and stitched on two sides to a polyester carrier was washed together in a launder-O-meter with 2.5 g of cotton fabric and 5 g/L of the liquid test laundry detergent, Table 4.

The conditions were as follows: Device: Launder-0-Meter from SDL Atlas, Rock Hill, USA.

Washing liquor: 250 ml, washing time: 60 minutes, washing temperature: 30° C. Water hardness: 2.5 mmol/L; Ca:Mg:HCO₃ 4:1:8

Fabric to liquor ratio 1:12 After the wash cycle, the multi stain monitors were rinsed in water, followed by drying at ambient temperature over a time period of 14 hours.

The following pre-soiled test fabrics were used:

CFT C-S-10: butter on cotton

CFT C-S-62: lard, colored on cotton

CFT C-S-68

EMPA 112: cocoa on cotton

EMPA 141/1: lipstick on cotton

EMPA 125:

wfk20D: pigment and sebum-type fat on polyester/cotton mixed fabric

CFT C-S-70: chocolate, mousse cream

wfk=wfk test fabrics GmbH, Krefeld

EMPA=Swiss Federal Institute of Materials Testing

CFT=Center for Test Material B.V.

The total level of cleaning was evaluated using color measurements. Reflectance values of the stains on the monitors were measured using a sphere reflectance spectrometer (SF 500 type from Datacolor, USA, wavelength range 360-700 nm, optical geometry d/8°) with a UV cutoff filter at 460 nm. In this case, with the aid of the CIE-Lab color space classification, the brightness L*, the value a* on the red-green color axis and the b* value on the yellow-blue color axis, were measured before and after washing and averaged for the 8 stains of the monitor. The change of the color value (A E) value, defined and calculated automatically by the evaluation color tools on the following equation:

Δ E=ΔDelta a*2+Δ Delta b*2++Δ Delta L*2,

Δ E is a measure of the achieved cleaning effect. All measurements were repeated six times to yield an average number. Note that higher A E values show better cleaning. A difference of 1 unit can be detected by a skilled person. A non-expert can detect 2 units easily. The results are shown in Table 5

R_(w)=washed soil reflectance

R_(o)=unsoiled reflectance

The increase in detergency due to the builder was calculated as: A total of 6 replications of each cloth were run during this study; a statistical confidence level of 90-95% was calculated.

Test formulations were manufactured by making formulations VII to XIII by mixing the components according to Table 4.

The respective salt (A) or comparative compound was added, if applicable, to the respective base formulation in amounts provided in Table 4.

Lipolase® 100 L was added, if applicable, to the respective base formulation in amounts provided in Table 4.

Savinase® 16.01 L was added, if applicable, to the respective base formulation in amounts provided in Table 4.

Water was added to accomplish the balance to 100.

TABLE 4 liquid laundry formulations Wt-% in formularion Ingredients VII VIII IX X XI XII XIII (B.1) 8 8 8 35 35 35 35 (B.2) 6 6 6 — — — — (B.3) 4 4 4 8 8 8 8 (B.4) — — — 10 10 10 10 (B.5) 4 4 4 4 4 4 4 (B.6) 2.5 2.5 2.5 — — — — Sorbitol — — — 2 2 2 2 PEI-EO20 5 5 5 5 5 5 5 Propyleneglycol 4 4 4 8 8 8 8 Glycerol — — — — — — — Ca-formiate — — — 2 2 2 2 Savinase 16.0/L* — — — — — 0.5 0,5 Lipolase* — — 0.4 — 0.4 0.4 0.4 (A) — 2.5 2.5 — 2.5 2.5 4 balance Water to 100 (B.1): n-C₁₈-alkyl-(OCH₂CH₂)₂₅—OH (B.2): C₁₀-C₁₈-alkylpolygycoside blend (B.3): Sodium C₁₀-C₁₂-alkyl benzenesulfonate (B.4): Sodium cumenesulfonate (B.5): Sodium laurethsulfate —n-C₁₂H₂₅—O—(CH₂CH₂O)₃—SO₃Na (B.6): n-C₁₂H₂₅(CH₃)₂N→O The Lipolase was present before storage in an amount of 7500 LU/ml The increase in detergency due to salt (A) was calculated as: a total of 6 examples of each cloth were run during this study; a statistical confidence level of >90% was calculated. Table 5 shows the sum of ΔE of the above mentioned multi-stain monitor. The launder-O-meter tests were executed with freshly prepared formulation (to) and with storing at 37° C. during a 2-month storage temperature. As an approximation one week at 37° C. is equivalent to 3½ weeks at 20° C.

TABLE 5 Results of launder-O-meter tests Formulation, (A) ΔE t0 ΔE1 week ΔE2 weeks ΔE4 weeks ΔE6 weeks ΔE8 weeks C-VII.0 nil 158 157 159 158 158 156 C- C-(A.21) 161 160 159 158 160 159 VIII.21 VIII.12 (A.12) 157 160 157 158 158 157 VIII.4 (A.4) 162 163 161 163 161 161 VII.2 (A.2) 160 159 161 158 160 158 IX.2 (A.2) 184 183 180 178 177 173 IX.3 (A.3) 183 184 181 179 179 175 IX.4 (A.4) 185 185 183 181 182 181 IX.7 (A.7) 181 179 180 178 179 177 C-X.0 nil 164 164 163 162 163 163 XI.5 (A.5) 191 189 188 188 184 185 XI.10 (A.10) 185 187 187 185 182 180 XI.12 (A.12) 185 186 186 187 185 185 XII.12 (A.12) 190 189 188 188 188 184 XII.2 (A.2) 191 189 186 186 184 182 XII.5 (A.5) 191 194 193 191 189 188 C- C-(A.15) 190 189 185 175 168 163 XIII.15 XIII.8 (A.8) 191 191 189 189 186 186 XIII.10 (A.10) 192 188 189 188 185 183 XIII.12 (A.12) 190 191 190 188 187 188 For comparative tests without salts (A) the latter were replaced by the same amount of diethylene glycol. 

1-15. (canceled) 16: A composition, comprising: (A) at least one salt of formula (I) (R²)₃N⁺—(CH₂)C(R³)(R⁴)—(O—X)_(m)—O—C(O)—R¹ A⁻  (I) wherein n is selected from 1 to 12, m is selected from 0 to 50, R¹ is selected from C₁-C₁₀-alkyl, linear or branched, and C₆-C₁₀-aryl, wherein R¹ may comprise one or more hydroxyl or C═O or COOH groups, partially or fully neutralized, if applicable, each R² is independently selected from C₁-C₁₀-alkyl and phenyl, R³ and R⁴ are each independently selected from hydrogen and C₁-C₄-alkyl, X is C₂-C₄-alkylen, and A⁻ is an organic counteranion; and (B) at least one surfactant selected from amphoteric and anionic and non-ionic surfactants. 17: The composition according to claim 16, wherein the at least one salt (A) of formula (I) comprises a counterion selected from tartrate, citrate, lactate and methanesulfonate. 18: The composition according to claim 16, which further comprises: (C) at least one enzyme. 19: The composition according to claim 18, wherein the at least one enzyme (C) is selected from proteases, amylases and lipases. 20: The composition according to claim 16, which is liquid at room temperature. 21: The composition according to claim 16, wherein each R² in the at least one salt (A) of formula (I) is methyl. 22: The composition according to claim 16, which comprises 0.5 to 30% by weight of the at least one salt (A) of formula (I). 23: The composition according to claim 16, which comprises a compound (A′) of formula (II) (R²)₃N⁺—(CH₂)_(n)C(R³)(R⁴)—(O—X)_(m)—OH R¹—COO⁻  (II) wherein R¹, R², X, n and m are the same as in the corresponding at least one salt (A) of formula (I). 24: The composition according to claim 16, wherein R¹ is selected from non-substituted C₁-C₆-alkyl, substituted C₁-C₁₀-alkyl, C₆H₅, para-HO—C₆H₄—, o,p-dihydroxyphenyl and 3,4,5-trihydroxyphenyl. 25: A method of laundering a fabric, the method comprising contacting the fabric with the composition of claim
 16. 26: A method of stabilizing an enzyme, the method comprising contacting the enzyme with the composition of claim
 16. 27: A process for making a composition, said process comprising: (a) mixing (A) at least one salt (A) of formula (I), and (C) at least one enzyme (R²)₃N⁺—(CH₂)_(n)C(R³)(R⁴)—(O—X)_(m)—O—C(O)—R¹ A⁻  (I) wherein n is selected from 1 to 12, m is selected from 0 to 50, R¹ is selected from C₁-C₁₀-alkyl, linear or branched, and C₆-C₁₀-aryl, wherein R¹ may comprise one or more hydroxyl or C═O or COOH groups, partially or fully neutralized, if applicable, each R² is independently selected from C₁-C₁₀-alkyl and phenyl, R³ and R⁴ are each independently selected from hydrogen and C₁-C₄-alkyl, X is C₂-C₄-alkylen, and A⁻ is an organic counteranion, and (b) adding at least one surfactant (B) selected from amphoteric and anionic and non-ionic surfactants. 28: The process according to claim 27, wherein the at least one salt (A) of formula (1) comprises a counterion selected from tartrate, citrate, lactate and methanesulfonate. 29: The process according to claim 27, wherein the at least one enzyme (C) is selected from proteases, amylases and lipases. 30: The process according to claim 27, wherein each R² in the at least one salt (A) of formula (I) is methyl. 31: A salt of formula (II) (CH₃)₃N⁺—(CH₂)CH(R³)—O—C(O)—R⁵ (A¹)⁻  (II) wherein (A¹)⁻ is selected from tartrate and citrate, R³ is selected from hydrogen and C₁-C₄-alkyl, and R⁵ is selected from —CH₂—C(OH)(COOX²)—CH₂—COOX² and —CH(OH)—CH(OH)—COOX¹, wherein X¹ is selected from hydrogen, alkali metal and (CH₃)₃N⁺—(CH₂)₂—, and each X² is independently selected from hydrogen, alkali metal and (CH₃)₃N′—(CH₂)₂—. 